Comparison of the Simple Cyclic Voltammetry (CV) and DPPH ... - MDPI
Molecules 2012, 17, 5126-5138; doi:10.3390/molecules17055126 OPEN ACCESS
molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article
Comparison of the Simple Cyclic Voltammetry (CV) and DPPH Assays for the Determination of Antioxidant Capacity of Active Principles Jesús F. Arteaga 1,*, Mercedes Ruiz-Montoya 1, Alberto Palma 1, Gema Alonso-Garrido 1, Sara Pintado 2 and JoséM. Rodríguez-Mellado 2 1
2
Department of Chemical Engineering, Physical Chemistry and Organic Chemistry, University of Huelva, Campus de El Carmen, 21071 Huelva, Spain Department of Physical Chemistry and Applied Thermodynamic, Córdoba University, Campus de Excelencia Internacional Agroalimentario (CEIA3), ed. Marie Curie, 14014 Córdoba, Spain
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +34-959-219999; Fax: +34-959-219983. Received: 2 March 2012; in revised form: 12 April 2012 / Accepted: 25 April 2012 / Published: 3 May 2012
Abstract: Antioxidant activity of a number of small (low molecular weight) natural compounds found in spices, condiments or drugs (gallic acid, sesamol, eugenol, thymol, carvacrol, vanillin, salicylaldehyde, limonene, geraniol, 4-hexylresorcinol, etc.) has been evaluated using electrochemical and DPPH• radical scavenging measurements. Structural analysis of the tested compound suggest a remarkable activity for phenol derivatives and the importance of the –R groups located on the phenolic ring in the molecule’s ability to act as free radical scavenging as well as their influence in the electrochemical behavior. The voltammetric method can be used for the determination of the antioxidant capability in the same manner as the DPPH• radical scavenging because of the correlation found between oxidation potentials and anti-radical power (ARP = 1/EC50). Such electrochemical determination is fast and cheap and allows making measurements under a variety of experimental conditions. The accuracy of the electrochemical measurements is the same for all the compounds, irrespective of their scavenging activity, the opposite of what occurs in the DPPH• test. Keywords: antioxidant activity; cyclic voltammetry; DPPH; phenolics; low weight antioxidants
Molecules 2012, 17
5127
1. Introduction The search for and use of natural and dietary antioxidants is growing because the public’s perception of their safety versus synthetic analogues [1,2]. Since ancient times, spices and condiments have been considered indispensable in the culinary arts, and, in addition, they have been recognized for their physiological and medicinal properties, and their broad-spectrum of effectiveness [3,4]. The antioxidant capability of these compounds, specially phenols [5], towards free radicals normally produced by cell metabolism or in response to external factors is due to the scavenging of free radicals and reactive oxygen species (ROS), which are made inactive [6], thus avoiding or preventing degenerative disorders caused in humans by oxidations of nucleic acids, proteins or lipids [7,8]. Plant phenolics arguably deserve a special mention when one considers that the wide-ranging benefits they offer to plants and hence to other living organisms are essentially all a result of their inherent physicochemical properties bundled within the phenol functional group. Different assays have been used to evaluate the antioxidant activity of natural products [9,10], but a comparison of the results is very difficult because of the different experimental methods adopted. Antioxidant activities of pure compounds and plant extracts have been determined, among others, by an accelerated test [11,12], by using radical species such as ABTS+• [13] and DPPH• [14], by the ESR spin trapping technique and by measuring the oxygen consumption in a heterogeneous lipid/water emulsion with lipid oxidation initiated by metmyoglobin [15]. However, all these procedures present some drawbacks since they require the use of specific reagents and tedious and time consuming sample preparation. Electrochemical measurements have advantages for the determination of antioxidant activity [16] such as their use as a rapid proof of the antioxidant capacity of a lot of organics. The oxidation potentials measured by cyclic voltammetry (CV) have been used to compare the antioxidant strength of compounds such as phenolic acids, flavonoids, cinnamic acids, etc. [16–20], being the glassy carbon electrode (GCE) the more frequently used electrode. Low oxidation potentials are associated with a greater facility or strength of a given molecule for the electrodonation and, thus, to act as antioxidant. There are some papers in the literature testing antioxidant capacity by using electrochemical measurements. Cyclic voltammetry at the GCE has been successfully applied to analyze antioxidants present in wine [21], plant extracts [22], phenolic standards [16] and even human plasma [20]. In these studies the most used parameter was the oxidation potential on the GCE, but this parameter strongly depends on the mechanism of the electrode reaction. The main drawback of the CV assay is that it is properly used to effectively characterize the reducing ability and reversibility of compounds either pure or presents in a real matrix. This value cannot always be directly related with the antioxidant ability of the sample. Besides, in the case of natural samples, the presence of compounds such as sugars or natural polymers could hinder the experimental manipulation as well as interfere with the interpretation of the potential values obtained. There are other studies comparing the data measured by cyclic voltammetry with those obtained by other methods [17–20,23–25]. The correlations are not always good [17], especially when the voltammetric and DPPH• assays were compared [24]. This last method evaluates the antioxidant activity of a given compound or a complex matrix by reaction in methanolic solution with a stable radical, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH•), which has an unpaired valence electron at one atom of its nitrogen bridge [25], the decrease of the DPPH•
Molecules 2012, 17
5128
concentration is measured from the decrease of absorbance at a characteristic wavelength. The correlation between the structure of the low molecular weight antioxidants, especially phenolics, and its antioxidant activity is not well determined at present, being the subject of intense research. In this paper, the electrochemical behaviour and the antioxidant effectiveness of a number of bioactive compounds are evaluated comparatively by means of the DPPH• test and cyclic voltammetry. It is intended to establish a relation between antioxidant capacity and oxidation potential to substitute the time-consuming DPPH• test by a rapid voltammetric determination. The accumulation of data of this kind is expected to be useful for an improved understanding of the role and activity of organic molecules as antioxidants, and the article would benefit from the analysis of a much larger number of organic molecules of these types. 2. Results and Discussion The compounds studied, whose structures are given in Figure 1, are mainly active principles of spices, seasonings or drugs, belonging to the family of low molecular weight antioxidants that are aromatic phenolics and non-phenolics, or cyclic and acyclic non-aromatic compounds, whose activities are based on their reducing properties. Figure 1. Structures of most of the antioxidants studied. O
OH
OH
OH
HO
HO
O
OH
OH
OH
O
H O 2,5-dihydroxybenzaldehyde
H O 2,4-dihydroxybenzaldehyde
4
2-furanacetic acid
Cl
cinnamic acid OH O
HO
4-hexylresorcinol
O
O
O OH geraniol
thymol O H
O p-chloroanisole
menthone
H
O vanillin
OH 3-hydroxycoumarin
O HO
H cinnamaldehyde
OH salicylaldehyde
O
O O sesamol O OH
OH HO H
limonene OH O
O
HO
HO
carvacrol
H
OH OH
HO
coumarin
O
O
O
O
OH
ascorbic acid
gallic acid
eugenol
furfural
Molecules 2012, 17
5129 Figure 1. Cont. O
O
OH
O
O
O
furoic acid
pulegone
OH
menthol
1,4-cineol
OH 4-methoxybenzyl alcohol
The main experimental problem of this method arises from the determination of the steady state concentration, since even for antioxidant species having a fast kinetics, after 24 h the decrease of absorbance continues, though at a low rate, and so it is difficult to obtain reproducible measurements. It must be noted that low uncertainties in EC50 can cause important changes in the ARP values; the inaccuracies derived from these measurements may provoke alterations in the order of antioxidant activity for a given family of compounds. It was considered that the steady state was reached when the absorbance remained constant during at least 10 min in the uncertainty limits of the spectrophotometer, i.e. ±0.001 absorbance units. The results obtained are gathered in Table 1. Table 1. Values of ARP and electrochemical parameters of the voltammograms recorded for the compounds studied. Compound ARP [a] Ep,a [b]/mV Compound ARP [a] Ep,a [b]/mV gallic acid (GA) 12.5 274 2,4-dihydroxybenzaldehyde (2,4-BZ)
molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article
Comparison of the Simple Cyclic Voltammetry (CV) and DPPH Assays for the Determination of Antioxidant Capacity of Active Principles Jesús F. Arteaga 1,*, Mercedes Ruiz-Montoya 1, Alberto Palma 1, Gema Alonso-Garrido 1, Sara Pintado 2 and JoséM. Rodríguez-Mellado 2 1
2
Department of Chemical Engineering, Physical Chemistry and Organic Chemistry, University of Huelva, Campus de El Carmen, 21071 Huelva, Spain Department of Physical Chemistry and Applied Thermodynamic, Córdoba University, Campus de Excelencia Internacional Agroalimentario (CEIA3), ed. Marie Curie, 14014 Córdoba, Spain
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +34-959-219999; Fax: +34-959-219983. Received: 2 March 2012; in revised form: 12 April 2012 / Accepted: 25 April 2012 / Published: 3 May 2012
Abstract: Antioxidant activity of a number of small (low molecular weight) natural compounds found in spices, condiments or drugs (gallic acid, sesamol, eugenol, thymol, carvacrol, vanillin, salicylaldehyde, limonene, geraniol, 4-hexylresorcinol, etc.) has been evaluated using electrochemical and DPPH• radical scavenging measurements. Structural analysis of the tested compound suggest a remarkable activity for phenol derivatives and the importance of the –R groups located on the phenolic ring in the molecule’s ability to act as free radical scavenging as well as their influence in the electrochemical behavior. The voltammetric method can be used for the determination of the antioxidant capability in the same manner as the DPPH• radical scavenging because of the correlation found between oxidation potentials and anti-radical power (ARP = 1/EC50). Such electrochemical determination is fast and cheap and allows making measurements under a variety of experimental conditions. The accuracy of the electrochemical measurements is the same for all the compounds, irrespective of their scavenging activity, the opposite of what occurs in the DPPH• test. Keywords: antioxidant activity; cyclic voltammetry; DPPH; phenolics; low weight antioxidants
Molecules 2012, 17
5127
1. Introduction The search for and use of natural and dietary antioxidants is growing because the public’s perception of their safety versus synthetic analogues [1,2]. Since ancient times, spices and condiments have been considered indispensable in the culinary arts, and, in addition, they have been recognized for their physiological and medicinal properties, and their broad-spectrum of effectiveness [3,4]. The antioxidant capability of these compounds, specially phenols [5], towards free radicals normally produced by cell metabolism or in response to external factors is due to the scavenging of free radicals and reactive oxygen species (ROS), which are made inactive [6], thus avoiding or preventing degenerative disorders caused in humans by oxidations of nucleic acids, proteins or lipids [7,8]. Plant phenolics arguably deserve a special mention when one considers that the wide-ranging benefits they offer to plants and hence to other living organisms are essentially all a result of their inherent physicochemical properties bundled within the phenol functional group. Different assays have been used to evaluate the antioxidant activity of natural products [9,10], but a comparison of the results is very difficult because of the different experimental methods adopted. Antioxidant activities of pure compounds and plant extracts have been determined, among others, by an accelerated test [11,12], by using radical species such as ABTS+• [13] and DPPH• [14], by the ESR spin trapping technique and by measuring the oxygen consumption in a heterogeneous lipid/water emulsion with lipid oxidation initiated by metmyoglobin [15]. However, all these procedures present some drawbacks since they require the use of specific reagents and tedious and time consuming sample preparation. Electrochemical measurements have advantages for the determination of antioxidant activity [16] such as their use as a rapid proof of the antioxidant capacity of a lot of organics. The oxidation potentials measured by cyclic voltammetry (CV) have been used to compare the antioxidant strength of compounds such as phenolic acids, flavonoids, cinnamic acids, etc. [16–20], being the glassy carbon electrode (GCE) the more frequently used electrode. Low oxidation potentials are associated with a greater facility or strength of a given molecule for the electrodonation and, thus, to act as antioxidant. There are some papers in the literature testing antioxidant capacity by using electrochemical measurements. Cyclic voltammetry at the GCE has been successfully applied to analyze antioxidants present in wine [21], plant extracts [22], phenolic standards [16] and even human plasma [20]. In these studies the most used parameter was the oxidation potential on the GCE, but this parameter strongly depends on the mechanism of the electrode reaction. The main drawback of the CV assay is that it is properly used to effectively characterize the reducing ability and reversibility of compounds either pure or presents in a real matrix. This value cannot always be directly related with the antioxidant ability of the sample. Besides, in the case of natural samples, the presence of compounds such as sugars or natural polymers could hinder the experimental manipulation as well as interfere with the interpretation of the potential values obtained. There are other studies comparing the data measured by cyclic voltammetry with those obtained by other methods [17–20,23–25]. The correlations are not always good [17], especially when the voltammetric and DPPH• assays were compared [24]. This last method evaluates the antioxidant activity of a given compound or a complex matrix by reaction in methanolic solution with a stable radical, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH•), which has an unpaired valence electron at one atom of its nitrogen bridge [25], the decrease of the DPPH•
Molecules 2012, 17
5128
concentration is measured from the decrease of absorbance at a characteristic wavelength. The correlation between the structure of the low molecular weight antioxidants, especially phenolics, and its antioxidant activity is not well determined at present, being the subject of intense research. In this paper, the electrochemical behaviour and the antioxidant effectiveness of a number of bioactive compounds are evaluated comparatively by means of the DPPH• test and cyclic voltammetry. It is intended to establish a relation between antioxidant capacity and oxidation potential to substitute the time-consuming DPPH• test by a rapid voltammetric determination. The accumulation of data of this kind is expected to be useful for an improved understanding of the role and activity of organic molecules as antioxidants, and the article would benefit from the analysis of a much larger number of organic molecules of these types. 2. Results and Discussion The compounds studied, whose structures are given in Figure 1, are mainly active principles of spices, seasonings or drugs, belonging to the family of low molecular weight antioxidants that are aromatic phenolics and non-phenolics, or cyclic and acyclic non-aromatic compounds, whose activities are based on their reducing properties. Figure 1. Structures of most of the antioxidants studied. O
OH
OH
OH
HO
HO
O
OH
OH
OH
O
H O 2,5-dihydroxybenzaldehyde
H O 2,4-dihydroxybenzaldehyde
4
2-furanacetic acid
Cl
cinnamic acid OH O
HO
4-hexylresorcinol
O
O
O OH geraniol
thymol O H
O p-chloroanisole
menthone
H
O vanillin
OH 3-hydroxycoumarin
O HO
H cinnamaldehyde
OH salicylaldehyde
O
O O sesamol O OH
OH HO H
limonene OH O
O
HO
HO
carvacrol
H
OH OH
HO
coumarin
O
O
O
O
OH
ascorbic acid
gallic acid
eugenol
furfural
Molecules 2012, 17
5129 Figure 1. Cont. O
O
OH
O
O
O
furoic acid
pulegone
OH
menthol
1,4-cineol
OH 4-methoxybenzyl alcohol
The main experimental problem of this method arises from the determination of the steady state concentration, since even for antioxidant species having a fast kinetics, after 24 h the decrease of absorbance continues, though at a low rate, and so it is difficult to obtain reproducible measurements. It must be noted that low uncertainties in EC50 can cause important changes in the ARP values; the inaccuracies derived from these measurements may provoke alterations in the order of antioxidant activity for a given family of compounds. It was considered that the steady state was reached when the absorbance remained constant during at least 10 min in the uncertainty limits of the spectrophotometer, i.e. ±0.001 absorbance units. The results obtained are gathered in Table 1. Table 1. Values of ARP and electrochemical parameters of the voltammograms recorded for the compounds studied. Compound ARP [a] Ep,a [b]/mV Compound ARP [a] Ep,a [b]/mV gallic acid (GA) 12.5 274 2,4-dihydroxybenzaldehyde (2,4-BZ)
